The present invention relates to a fermentation process for producing mannitol from fructose using a novel strain, Candida magnoliae (KCCM-10252) producing mannitol in high yield and high productivity, and a novel strain of Candida magnoliae (KCCM-10252) which is isolated from a natural substance.
Mannitol is a six carbon sugar alcohol, and it is contained in natural substances, such as, brown algae, mushrooms, fungi and the like in nature. Since it has 30xcx9c40% of sugar""s sweetness, not only it can be used as an alternative sweetening agent in the food processing industry where the use of sugar is limited, but also it can be mostly used as an additive for confectionary, a filler for medicine, a surfactant, a waterproof agent and the like, because it has superior properties, such as, a cool taste, low hygroscopic property, fluidity and the like. Further, it has been widely used in the food and pharmaceutical industry, for example, as a therapeutic intermediate of hypotension and a coating agent for reducing a bitter taste during the manufacturing various drugs.
Mannitol exists in many kinds of fruits or vegetables as a natural product in a very small amount. Therefore, it is industrially of little economical value to extract mannitol from fruits or vegetables.
One of the commercialized methods for producing mannitol comprises i) isolating the fructose formed by hydrolysis from sugar and the like ; and ii) manufacturing the mannitol by hydrogenation to the fructose in the presence of a catalyst under high temperature and high pressure. However, since the sorbitol is produced as a by-product in such manufacturing process, it is required to purify the mannitol in order to eliminate the sorbitol as a separate step. Of course, the conversion yield is so much low that its production cost becomes high. Further, such manufacturing process has handicaps, such as, a risk of dangerousness and requirement of wastes treatment, because it includes the reaction under high temperature and high pressure.
In order to solve such drawbacks, many studies on a method for producing mannitol by using microorganisms have been conducted. As the known microorganisms related with production of mannitol, in case of any yeast, there are Candida zeylannoide, Candida lipolitica, Cryptococcus neoformans, Torulopsis mannitofaciens, etc.,; in case of bacteria, there are Lactobacillus brevis, Leuconostoc mesenteriode, Mycobacterium smegmatis, etc.,; and in case of fungi, there are Mucor rouxii, Aspergillus nidulans, Penicillum scabrosum, etc. The method for producing mannitol by using microorganisms has a high economical value. Further, since only mannitol can be specifically produced from glucose or fructose according to the method, the process for isolating and purifying mannitol after completion of the fermentation for mannitol can be performed with much convenience. However, it is difficult to industrialize this method, because its productivity and yield is not so high.
It is, therefore, an objective of the present invention to provide a method for producing mannitol in high productivity and high yield by isolating, identifying and developing a new strain, Candida magnoliae producing mannitol in high yield from a fermentation sludge collected from xylitol manufacturing company (Bolak Co., Ltd., Osan, Korea) and further optimizing culture conditions.
The object of the present invention is to provide novel strain of Candida magnoliae, which was deposited Korea Culture Center of Microorganism, 361-221, Yurim B/D Hongje-1-dong, Seodaemun-gu, Seoul, 120-091, Republic of Korea with accession number KCCM-10252 on Mar. 5, 2001 under Budapest treaty, for preparing mannitol with high yield and high productivity.
Another object of the present invention is to provide the fermentation process for manufacturing mannitol with high yield and high productivity, using strain of Candida magnoliae (KCCM-10252) comprising the steps of: i) fermenting glucose or fructose medium with microorganism by controlling following fermentation conditions; a) composition of medium for maximum production of mannitol consists of 50xcx9c100 g/L of glucose, 50xcx9c120 g/L of fructose, 5xcx9c20 g/L of yeast extract, 1xcx9c5 g/L of ammonium sulfate, 1.0xcx9c5.0 g/L of dibasic potassium phosphate and 0.1xcx9c0.5 g/L of magnesium sulfate; b) pH of culture medium is 4.5xcx9c5.5; c) temperature of cultivation is 27xcx9c35xc2x0 C.; d) aeration rate is 0xcx9c0.5 volume of air per volume of medium per minute; and e) agitation speed of the medium is 200xcx9c500 rpm; ii ) removing the cells and other residue from the fermentation medium; and iii) separating and recovering mannitol from the fermentation medium of step ii).
The further object of the present invention is to provide a fermentation process wherein the strain used for fermentation is prepared by cultivating frozen Candida magnoliae (KCCM-10252) in YPD medium comprising 15xcx9c25 g/L of glucose, 7xcx9c13 g/L of yeast extract and 15xcx9c25 g/L of peptone.
Also, the present Invention is to provide an isolating method for novel strain, Candida magnoliae (KCCM-10252), comprising steps of i) sorting a single colony growing fast at 27xcx9c33xc2x0 C. by using a plate containing 10xcx9c20 g/L of glucose, 18xcx9c22 g/L of fructose, 15xcx9c25 g/L of peptone, 7xcx9c15 g/L of yeast extract and 12xcx9c17 g/L of agar after diluting a natural sludge sample; ii) sorting the strain producing mannitol most after fermenting such sorted strains in the medium containing 30xcx9c70 g/L of glucose, 50xcx9c100 g/L of fructose, 5xcx9c20 g/L of yeast extract, 1xcx9c5 g/L of ammonium sulfate and 1xcx9c5 g/L of dibasic potassium phosphate for 68xcx9c76 hours, and iii) isolating the novel strain, Candida magnoliae (KCCM-10252) producing the mannitol.
The method for isolating the new strain, Candida magnoliae according to the present Invention is described as follows.
After properly diluting a fermentation sludge sample collected from xylitol manufacturing company, a single colony with priority given to strains growing fast at 27xcx9c33xc2x0 C. was sorted by using the plate containing 200xcx9c400 g/L of glucose, 15xcx9c25 g/L of peptones, 7xcx9c15 g/L of yeast extract and 12xcx9c17 g/L of agar, and further the strain producing mannitol most was finally sorted after such sorted strains were fermented in the medium containing 30xcx9c70 g/L of glucose, 50xcx9c100 g/L of fructose, 5xcx9c20 g/L of yeast extract, 1xcx9c5 g/L of ammonium sulfate and 1xcx9c5 g/L of dibasic potassium phosphate for 68xcx9c76 hours.
For identification of the strain, the method (Kurtzman and Robnett, 1998) for analyzing the nucleotide sequence of 26s rRNA and the morphological, cultural, and physiological properties thereof were examined in accordance with Yarrow""s method (Elsevier Science Publishers, Amsterdam, 1998) and further the strain was identified according to the classification method of Barnett, et al.(Yeasts: Characteristics and identification. Cambridge University Press, London, 1983). The results of analyzing the nucleotide sequence of 26s rRNA of the sorted strain and the nucleotide sequence of similar seeds are shown in Table 1 and Table 2, respectively.
Furthermore, the morphological, physiological and biochemical properties thereof are shown in Table 3. The available carbon source for the new strain is shown in Table 4.
As a result of identifying properties of the strain mentioned above, the strain proved to be of the Candida magnoliae seed. Further, the novel strain, Candida magnoliae was deposited with Korean Culture Center of Microorganisms on Mar. 5, 2001 (KCCM-10252).
The method for producing mannitol by fermentation according to the present Invention is more specifically described as follows.
The YPD medium containing 15xcx9c25 g/L of glucose, 7xcx9c13 g/L of yeast extract and 15xcx9c25 g/L of peptone was used as a growth medium for seed culture of the strain. The medium comprising 50 g/L of glucose and 100 g/L of fructose as the carbon source, yeast extract and ammonium sulfate as the nitrogen source and dibasic potassium phosphate and magnesium sulfate as inorganic salts was used as a fermentation medium of the flask. Each component""s quantity may be changed in order to increase productivity of mannitol. The medium used in the fermenter was an optimum medium comprising 300 g/L (concentration of a total added quantity) of fructose, 50 g/L of glucose, 10 g/L of a yeast extract, 2.5 g/L of dibasic potassium phosphate, 2.5 g/L of ammonium sulfate and 0.4 g/L of magnesium sulfate.
The seed culture was performed by inoculating the strain kept in a cold room with 50 ml of the YPD medium contained in the 250 ml flask until the strain grew in the shaking culture medium at 230xcx9c270 rpm and 27xcx9c33xc2x0 C. (for about 24 hours) so much as the concentration of the biomass thereof reached to 3xcx9c4 g/L. The flask culture was performed by inoculating the seed culture fluid equivalent to 5% of the medium with 50 ml of the fermentation medium contained in the 250 ml flask and it was cultured in the shaking culture medium at 230xcx9c270 rpm and 27xcx9c33xc2x0 C. for 68xcx9c78 hours.
For the fed-batch fermenter culture, the 5L fermenter (Korea Fermenter Co., Ltd.) having 2L of the medium in which 100 g/L of fructose was contained at the initial fermentation stage was used, and the stirring rate and the ventilation volume were adjusted to 200xcx9c500 rpm and 0xcx9c0.5 vvm respectively in the fermentation process. The pH was adjusted to 4.5xcx9c5.5 in the pre-fermentation process, the culture temperature was 27xcx9c33xc2x0 C., and the concentration of the maintained fructose was adjusted to 50xcx9c120 g/L.
The respective concentrations of glucose, fructose and mannitol were measured by using HPLC""s Refractive Index Detector (Waters 2410, USA) having Carbohydrate Analysis Column (Waters, USA) equipped therewith. Wherein, a mixture of acetonitrile and water(8:2) was used as a solvent, and the flow rate thereof was 1.5 ml/min. For obtaining the concentration of biomass thereof, the suspension degree thereof was measured at 600 nm by using the turbidimeter and further it was converted into a dry-weight by using the standard curve as measured beforehand. The concentration of the dissolved oxygen was measured by using the dissolved oxygen electrodes of Ingold Company (Swiss, polarographic type).